Most of immunoadjuvants widely used so far have two kinds of roles at a local administration site in vivo, specifically, (1) gradually releasing antigens to efficiently supply them to immunocompetent cells with maintaining the antigens in a state of aqueous or oil emulsion to prevent rapid disperse of the antigens, and (2) inducing inflammatory reactions and activating the immunocompetent cells gathering at the local inflammation site. In recent years, it has been revealed that antigen-presenting cells play a major role of immune responses in both of the humoral immune responses inducing antibody production and the cell-mediated immune responses inducing killer T cells, and that dendritic cells, macrophages, and B cells exist as the antigen-presenting cells. Among them, the dendritic cells have the most potent antigen-presenting ability (Dendritic Cells, Second edition, ed. by Lotze, M. T. and Thomson, A. W., Academic Press, San Diego, 2001). When a substance that can effectively activate antigen-presenting cells is administered to a living body together with an antigen, the substance acts as an immunoadjuvant and can efficiently induce and enhance immune responses against the antigen in both of the humoral immune responses and cell-mediated immune responses.
Among the variety of conventionally known immunoadjuvants, only a few immunoadjuvants are safe enough to be usable in a tumor immunotherapy for a purpose of therapeutic treatment, and prevention of metastasis and recurrence of human tumors, and also are inexpensive. For example, keyhole limpet hemocianin (KLH) has been used as an immunoadjuvant in a tumor immunotherapy by using cultured dendritic cells (Geiger, J. D., et al., Cancer Res., 61:8513-8519, 2001). However, this substance is expensive. Methods of directly administering cytokines such as granulocyte-macrophage colony stimulating factor (henceforth also abbreviated as “GM-CSF”), which directly activate dendritic cells, as immunoadjuvants have also been proposed. However, cytokines are still much more expensive.
As safe and inexpensive immunoadjuvants for manufacture of vaccines as a measure against infectious diseases, immunoadjuvants having insufficient activity such as alum (aluminum hydroxide), Freund's incomplete adjuvant (since this adjuvant is oily substance, toxicity is concerned), and Montanide have been used. Although these substances are less toxic compared with Freund's complete adjuvant (henceforth also abbreviated as “FCA”) and Ribi adjuvant system, which are used in animal experiments, immunoadjuvant activities thereof are also weak.
Tuberculin for detection of Mycobacterium tuberculosis infection history, especially, tuberculin purified protein derivative (henceforth also abbreviated as “PPD”), which consists of protein ingredients in tuberculin purified by ammonium sulfate precipitation, is extremely safe even if repetitively administered to humans and moreover inexpensive. Therefore, tuberculin has been widespread all over the world. The inventors of the present invention found that PPD could be used as an immunoadjuvant (PCT/JP00/00692). The inventors of the present invention also found that, when PPD was formed as precipitate by coacervation with soluble proteins and mucopolysaccharides and then administered into tumor tissues denatured by a physical means, the PPD could served as an effective immunoadjuvant for inducing antitumor immune responses (Japanese Patent No. 3492671). This immunoadjuvant has a higher immunoadjuvant activity compared with dissolved PPD, and has extremely high safety in the same manner as the dissolved PPD. However, the immunoadjuvant activity thereof is insufficient if compared with lipopolysaccharides (henceforth also abbreviated as “LPS”) which are major ingredients of FCA or endotoxins.
In peripheral blood, immature antigen-presenting cells flow which can phagocytize microparticle antigens. It is known that when a lipopolysaccharide (LPS) is added to cultured immature antigen-presenting cells in vitro, maturation of the cells is advanced to exhibit potent ability to present antigens. The antigen-presenting cells activated in this process release various kinds of cytokines, such as GM-CSF, interleukin (henceforth also abbreviated as “IL”) 12, and interferon-γ (henceforth also abbreviated as “IFNg”). GM-CSF itself is essential as a cell growth factor of the dendritic cells. Therefore, the dendritic cells once activated become possible to maintain the activated state for a long period of time and continue to survive on the basis of the autocline mechanism of GM-CSF. Macrophages, as well as dendritic cells, are isogenic antigen-presenting cells which are differentiated from the same hematopoietic stem cells, and produce cytokines such as GM-CSF upon receipt of immunostimulation in the same manner as dendritic cell.
Practically, antitumor immune responses to tumor cells can be efficiently induced by administering solidified and microparticulated antigen tumor tissues as an antigen into a living body together with a cytokine such as GM-CSF (PCT/JP00/00692). This fact indicates that GM-CSF itself serves as an immunoadjuvant, and at the same time, an amount of GM-CSF produced from antigen-presenting cells stimulated with the immunoadjuvant can serve as an index representing the activity of the original immunoadjuvant. In other words, quantification of GM-CSF produced by antigen-presenting cells derived from a human enables successful estimation of immunoadjuvant activity in human in an in vitro experimental system without using human individuals.
By using peripheral blood adherent cells including antigen-presenting cells derived from human peripheral blood, the inventors of the present invention demonstrated, on the basis of the aforementioned means, that a potent immunoadjuvant action could be obtained by immobilizing soluble ingredients derived from microorganisms [soluble ingredients extracted with an organic solvent such as Mycobacterium bovis Bacillus Calmette-Guerin (henceforth also abbreviated as “BCG bacterium”)] on a solidified tissue as an immunostimulating substance carrier (WO2003/074079).
The solidified tissue used as the immunostimulating substance carrier in the aforementioned immunoadjuvant is a biodegradable material which is digested in phagocytes including antigen-presenting cells and disappears without remaining in the body. However, when this immunoadjuvant is applied to a human individual, if a tissue used is not from the individual himself wherein major histocompatibility antigen is completely identical, or a tissue of the other one of monozygotic twins wherein major histocompatibility antigen is genetically identical, the immunostimulating substance carrier may have antigenicity to the human individual, and may sometimes induce an antigen-antibody reaction against the immunostimulating substance carrier or digested protein fragments thereof. This causes a problem that, when the aforementioned immunoadjuvant is used as a general-purpose immunoadjuvant (when a desired antigen is added to the immunoadjuvant to induce an immune response to the antigen), undesired antigen-antibody reactions against the immunostimulating substance carrier itself are induced, and the immune response to the antigen is buried among the whole immune responses and become unlikely to be manifested.
The inventors of the present invention also proposed a method for preparing calcium phosphate microparticles carrying one kind or two or more kinds of molecules to be carried (Japanese Patent Unexamined Publication (KOKAI) No. 2005-126335). These calcium phosphate microparticles consist of hydroxyapatite having a Ca/P molar ratio of 1.3 or higher. It has been further demonstrated that since amorphous calcium phosphate microparticles and low-crystalline apatite can carry not only hydrophilic protein molecules but also hydrophobic low molecules, they can serve as an immunostimulating substance carrier (Japanese Patent Unexamined Publication (KOHYO) No. 2002-524491). However, any immunoadjuvant consisting of a mixture of two or more kinds of immunostimulating substance carriers having completely different physicochemical properties and carrying one kind or two or more kinds of immunostimulating substances have not been known so far.